The long range objective of this research is to study the relationship between the interactions of the cytoskeletal proteins of human platelets, their organization in the cell body, and their redistribution when platelets are stimulated, leading to such changes as filopodia extension, secretion, aggregation and clot retraction. The membrane-cytoskeleton complex no doubt acts as a vehicle for transmembrane communication whereby the binding of a ligand molecule to a surface receptor results in the movement and/or restructuring of the cytoskeleton on the cytoplasmic side of the membrane. Since the platelet cytoskeleton was first isolated in our laboratory we have devoted our studies to characterizing the relationship of the constituent proteins to each other and to their interaction with the plasma membrane. We have been particularly interested in the role that Ca2+, proteolysis and phosphorylation play in regulating these interactions and during the next grant period it is our intention to continue these studies in light our most recent information regarding the role of ABP phosphorylation in protecting ABP from proteolytic degradation by calpain and regulating its interaction with F- actin. Since both F-actin and ABP are presumably linked to the membrane via glycoprotein-ABP interaction, either directly or by means of an intermediary protein, the importance of these observations may relate to understanding cytoskeleton/membrane interactions and their role in mediating extracellular/intracellular signalling. To this end our specific aims will deal with a number of consequences of ABP phosphorylation, namely 1) sites of phosphorylation 2) conformational changes associated with phosphorylation 3) role of cAMP dependent kinase and calmodulin activated phosphatase in regulating ABP phosphorylation and 4) its interaction with membrane and F-actin. We will continue to investigate the F-actin attachment site to the membrane and the re- association of F-actin to the isolated plasma membrane. In light of the recent delineation of F-actin between two distinct regions of the platelet, namely a membrane skeleton and a cytoplasmic domain, our earlier description of two forms of platelet actin, takes on added significance. We will pursue structural studies on these two forms and further characterize their differences and localization within the platelet. Finally, we will investigate the role of a newly isolated "synexin" like protein from platelets which binds Ca2+ and may figure prominently in the interaction of F-acting to the platelet membrane.